The ability of HIV-1 to utilize the cellular machinery for nuclear entry allows the virus to infect resting CD4+ T cells and tissue macrophages, the two non-dividing cell types believed to play critical roles in HIV-1 transmission, persistence and pathogenesis. However, the precise mechanism and specific requirement of nuclear entry in HIV-1 replication remains unclear. Capsid is the major determinant for HIV-1 infection of non-dividing cells but also dictates the dependence of HIV-1 on TNPO3, a host molecule recently implicated in HIV-1 nuclear entry. Our preliminary studies found a perfect correlation between the dependence of HIV-1 capsid mutants on TNPO3 and their ability to infect non-dividing cells. The hypothesis being tested in this project is that the capsid protein regulates HIV-1 nuclear entry by assisting intracellular virus complexes in hijacking the TNPO3-dependent cellular machinery and this ability is critical for HIV-1 replication in vivo. In Aim 1, we will determine hw the HIV-1 capsid protein determines TNPO3 usage by HIV-1 and how TNPO3 and other host factors promote HIV-1 nuclear entry. We will next define the specific requirement of this TNPO3-dependent nuclear entry for HIV-1 replication in vivo. In Aim 2, we will study a unique case of HIV-1 capsid evolution in patients carrying the HLA-B27. Preliminary data suggests recurrent selection of TNPO3- dependent viruses in these patients. In Aim 3, we will combine our genetic data with a well-studied macaque model of HIV-1 by using simian immunodeficiency virus (SIV) to directly address the importance of nuclear entry in vivo. Here we will generate and utilize TNPO3-independent SIV mutants, which either retain or lose the ability to infect non-dividing cells. Successful completion of this proposed research will help us understand the mechanism by which HIV-1 exploits the nuclear transport machinery to maximize its propagation. PUBLIC HEALTH RELEVANCE: The ability of HIV-1, the causative agent of AIDS, to actively transport viral DNA into the nucleus expands its cellular range to non-dividing cell types such that HIV-1 can infect resting CD4+ T cells and tissue macrophages. The goal of this project is to determine the mechanism and role of nuclear entry in HIV-1 replication. We expect that our findings could have practical implications for the development of novel antiviral drugs targeting viral capsid protein and nuclear entry and will provide novel insights into how resting CD4+ T cells and macrophages contribute to multiple aspects of HIV-1 infection in vivo.